TAT/CT-1及TAT/EGFP融合蛋白的表达与纯化及其在小鼠体内分布的观察

目的分别构建含蛋白转导域(protein transduction domain,PTD)TAT与小鼠心肌营养素-1(CT-1)及TAT与增强绿色荧光蛋白(enhanced green fluorescent protein,EGFP)融合基因片断的原核表达质粒TAT/CT-1及TAT/EGFP,利用大肠杆菌进行表达并纯化;观察融合蛋白在小鼠体内的分布.方法采用PCR及克隆技术将CT-1编码区基因以及EGFP基因分别与TAT连接到原核表达载体pGEX-4T3上.用IPTG诱导表达融合蛋白,谷胱甘肽琼脂糖4B纯化融合蛋白.小鼠尾静脉注射融合蛋白,脑、脊髓、肝、心肌等器官冰冻切片,免疫荧光观察融合蛋白的存在.结果目的片断CT-1及EGFP被有效的扩增,构建后质粒测序表明无PCR引起的突变,TAT/CT-1及TAT/EGFP在转化的大肠杆菌中获得高效表达,并成功纯化.脑、脊髓、肝、心肌等组织切片免疫荧光检测呈阳性.结论成功获得了有活性的TAT/CT-1及TAT/EGFP的基因表达产物;TAT可介导CT-1及EGFP由细胞外跨膜转导进入细胞内;为CT-1功能的进一步研究打下了基础.

Expression and purification of TAT/CT-1 and TAT/EGFP fusion proteins

Objective To construct a vector containing protein transduction domain (PTD) and cardiotrophin-1 and a vector containing PTD and EGFP, express them in E. coli. and purify them. To detect the distribution of the two fusion proteins in mice. Methods CT-1 and EGFP were cloned to GST-fusion expression vector pGEX-4T3 by PCR and cloning techniques respectively, and then TAT was cloned into the vectors respectively to give pGEX-TAT/CT-1, pGEX-TAT/EGFP. After induced by IPTG the soluble protein GST-TAT/CT-1 and GST-TAT/EGFP was purified by Glutathione Sepharose 4B. The purified fusion proteins were injected into mice through caudal vein and examined in tissue section by immunohistochemical staining. Results CT-1 and EGFP were effectively amplified and the TAT/CT-1 and TAT/EGFP gene sequencing showed the same sequence as scheduled. The fusion proteins was successfully expressed in E. coli. and purified. Conclusion TAT/CT-1 and TAT/EGFP fusion proteins were expressed and purified successfully. The two fusion proteins were all detected positively in mouse brain, spinal cord, heart and liver.